吐温80是药物引起类过敏反应的重要原因，但机制不清。肥大细胞Mas相关G蛋白偶联受体（MRGPR）是类过敏反应的新靶点。我们推测吐温80可能通过MRGPR激活PLC/STIM1/TRPC通路诱导肥大细胞脱颗粒，引起类过敏反应。本项目拟在以往研究基础上，先用shRNA和质粒转染技术建立hMRGPRX2基因沉默LAD2细胞和hMRGPRX2/293T高表达细胞，研究该受体表达对吐温80引起的脱颗粒、凋亡和钙内流的影响；再用PLC、STIM1和TRPCsiRNA及通路抑制剂，研究吐温80对MRGPR介导的信号分子PLC、STIM1和TRPC等蛋白表达、TRPC电流及STIM1/TRPC交互作用等的影响；最后用mMrgprB2MUT小鼠研究吐温80对小鼠类过敏效应及信号分子的影响，阐明吐温80通过MRGPR介导的类过敏反应机制。项目的完成能为寻找抗类过敏药物作用靶点和敏感人群筛查指标提供理论依据。

Many drugs such as vitamin K1 injection and multivitamin injection can induce serious hypersensitivity reactions which attract extensive concerns. Our previous researches showed that the serious adverse reactions of vitamin K1 injection and multivitamin injection were anaphylactoid reaction caused by its solubilizer tween 80. As a commonly used pharmaceutic adjuvant, meanwhile, tween 80 is one of the most important causes resulting in anaphylactoid reaction induced by drugs. However, the mechanism of tween 80-induced anaphylactoid reaction remains unclear. MAS-related G protein coupled receptor (MRGPR) on mast cell is a new target of anaphylactoid reaction. It was reported that loss of hMRGPRX2 function in LAD2 mast cells abrogated Ca2+ mobilisation and histamine release when the cells were stimulated with basic secretagogues and the hMRGPRX2 agonists. Moreover, phospholipase C (PLC), stromal interaction molecule-1 (STIM1), and transient receptor potential channel (TRPC) are related to Ca2+ mobilization which leads to exocytosis of preformed granule-associated mediators in mast cell. Therefore, we speculate that tween 80 could activate the PLC/STIM1/TRPC pathway by acting on MRGPR, lead to Ca2+ mobilization, and then induce the degranulation of mast cell resulting in anaphylactoid reactions. Firstly, we will investigate the effect of the different level of hMRGPRX2 gene expression on the tween 80-induced degranulation of LAD2 cells. shRNA and plasmid transfection techniques will be used to establish hMRGPRX2 gene silent LAD2 cells and hMRGPRX2 gene overexpression cells, respectively. The changes of degranulation, cell apoptosis, and Ca2+ mobilisation will be detected by spectrophotometry, flow cytometry, and laser scanning confocal microscopy, respectively. Then, we will explore the regulatory role of PLC/STIM1/TRPC pathway in hMRGPRX2 mediated tween 80-induced anaphylactoid reaction. siRNA techniques and pathway inhibitor will be used to suppress the expression and effect of PLC, STIM1 and TRPC in LAD2 cells, respectively. The mRNA and protein expressions of molecules including PLC, STIM1, and TRPC will be detected by qRT-PCR and western blotting. The TRPC channel current and the interaction effect of STIM1 and TRPC will be tested by patch clamp single channel current recording technology, multifluorescent label/confocal and co-immunoprecipitation method. Finally, mMrgprB2MUT mice will be used to research the anaphylactoid reaction induced by tween 80 in vivo by detecting the change of the plasma consentration of histamine and trypsase, and the level of molecules in this pathway. The findings will unveil the mechanisms of tween 80-induced anaphylactoid reaction, which will provide theoretical basis for developing novel drugs against anaphylactoid reaction and finding examination indicator to screen sensitive population. The project has an important significance for prevention and treatment of anaphylactoid reaction.